Limits...
Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector.

Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK - BMC Microbiol. (2009)

Bottom Line: The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes.To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

View Article: PubMed Central - HTML - PubMed

Affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), ICGEB Campus, Aruna Asaf Ali Marg, New Delhi, India. asharani_verma@yahoo.co.in

ABSTRACT

Background: Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.

Results: Lab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74-2.14 for lab-reared and in the range of 2.75-3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.

Conclusion: More than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

Show MeSH

Related in: MedlinePlus

Percentage abundance diagram of culturable isolates and 16S rRNA gene library clones from lab-reared (LR) and field-collected (FC) adult male, female and larvae of Anopheles stephensi. Percentage distribution was calculated on the basis of relative abundance in the total PCR amplification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2698833&req=5

Figure 1: Percentage abundance diagram of culturable isolates and 16S rRNA gene library clones from lab-reared (LR) and field-collected (FC) adult male, female and larvae of Anopheles stephensi. Percentage distribution was calculated on the basis of relative abundance in the total PCR amplification.

Mentions: Out of a total of 50 screened bacterial colonies, 34 distinct isolates, 18 from adult male and 16 from adult female lab-reared A. stephensi were studied further. 16S rRNA sequencing placed these two sets of 18 and 16 isolates with their closest matches into 4 major groups. In lab- reared adult male A. stephensi isolates, 3 major groups were: Cytophaga-flavobacter-bacteroidetes (CFB), alphaproteobacteria and gammaproteobacteria, whereas in lab-reared adult female betaproteobacteria was also identified (Figure 1). 16S rRNA gene sequence identified the lab-reared adult male bacterial isolates as Agrobacterium sp., Chryseobacterium meninqosepticum, Pseudomonas mendocina and Serratia marcescens, whereas in lab-reared A. stephensi adult female Comamonas sp. was also present, the details of which are shown in Table 1. In lab-reared adult male and female A. stephensi, most abundant and diverse members were of gammaproteobacteria (61% and 43% respectively) particularly, Pseudomonas mendocina and S. marcescens, as a dominant group. It was followed by CFB group bacteria (Chryseobacterium meninqosepticum) constituting around 33% and 38% in male and female A. stephensi, respectively. Distinctive representative genera in lab-reared female A. stephensi was Comamonas sp. (betaproteobacterium), representing 13% of total isolates. However, male A. stephensi isolates were distinguishable by genera such as Agrobacterium sp., an alphaproteobacterium. Chryseobacterium, Pseudomonas and Serratia were genera common to adult male and female A. stephensi.


Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector.

Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK - BMC Microbiol. (2009)

Percentage abundance diagram of culturable isolates and 16S rRNA gene library clones from lab-reared (LR) and field-collected (FC) adult male, female and larvae of Anopheles stephensi. Percentage distribution was calculated on the basis of relative abundance in the total PCR amplification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698833&req=5

Figure 1: Percentage abundance diagram of culturable isolates and 16S rRNA gene library clones from lab-reared (LR) and field-collected (FC) adult male, female and larvae of Anopheles stephensi. Percentage distribution was calculated on the basis of relative abundance in the total PCR amplification.
Mentions: Out of a total of 50 screened bacterial colonies, 34 distinct isolates, 18 from adult male and 16 from adult female lab-reared A. stephensi were studied further. 16S rRNA sequencing placed these two sets of 18 and 16 isolates with their closest matches into 4 major groups. In lab- reared adult male A. stephensi isolates, 3 major groups were: Cytophaga-flavobacter-bacteroidetes (CFB), alphaproteobacteria and gammaproteobacteria, whereas in lab-reared adult female betaproteobacteria was also identified (Figure 1). 16S rRNA gene sequence identified the lab-reared adult male bacterial isolates as Agrobacterium sp., Chryseobacterium meninqosepticum, Pseudomonas mendocina and Serratia marcescens, whereas in lab-reared A. stephensi adult female Comamonas sp. was also present, the details of which are shown in Table 1. In lab-reared adult male and female A. stephensi, most abundant and diverse members were of gammaproteobacteria (61% and 43% respectively) particularly, Pseudomonas mendocina and S. marcescens, as a dominant group. It was followed by CFB group bacteria (Chryseobacterium meninqosepticum) constituting around 33% and 38% in male and female A. stephensi, respectively. Distinctive representative genera in lab-reared female A. stephensi was Comamonas sp. (betaproteobacterium), representing 13% of total isolates. However, male A. stephensi isolates were distinguishable by genera such as Agrobacterium sp., an alphaproteobacterium. Chryseobacterium, Pseudomonas and Serratia were genera common to adult male and female A. stephensi.

Bottom Line: The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes.To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

View Article: PubMed Central - HTML - PubMed

Affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), ICGEB Campus, Aruna Asaf Ali Marg, New Delhi, India. asharani_verma@yahoo.co.in

ABSTRACT

Background: Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. Anopheles stephensi is one of the vectors of malaria parasite Plasmodium vivax. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about Anopheles-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.

Results: Lab-reared and field-collected A. stephensi male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught A. stephensi mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval A. stephensi, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared A. stephensi midgut, respectively. These OTUs had a Shannon diversity index value of 1.74-2.14 for lab-reared and in the range of 2.75-3.49 for field-caught A. stephensi mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male A. stephensi were uncultured Paenibacillaceae while in female and in larvae it was Serratia marcescens, on the other hand in lab-reared mosquitoes, Serratia marcescens and Cryseobacterium meninqosepticum bacteria were found to be abundant.

Conclusion: More than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of A. stephensi from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".

Show MeSH
Related in: MedlinePlus